Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R. capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1-lacZ and nifA2-lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen-deficient conditions. This nitrogen-controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON-activated promoters.
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PMID:Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN. 137 28

Two duplexes (20-mers) were constructed containing either a single cis-[Pt(NH3)2[d(GpG)]] or cis-[Pt(NH3)2[d(ApG)]] intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II). These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases. Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates. Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites. In particular, binding affinity between E. coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent Km of enzyme for the platinated polymer is increased by a factor of 4-5. In contrast, binding affinity between the RNA polymerase and the d(ApG)-containing template is not affected by modification of the d(ApG) site by cis-diamminedichloroplatinum(II). Similar experiments were carried out with synthetic templates containing the adducts at the d(GpG) sites, in which one of the two platinated dG residues is paired with a dT residue.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II). 153 34

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with the E. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.
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PMID:Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA. 166 78

Escherichia coli RNA polymerase (RNAP) exhibits a strong selectivity for the secondary structure of its template DNA, as shown by the influence both of the DNA conformation on the transcription cycle and of the enzyme on the DNA conformation itself. Binding, chain initiation and elongation characteristics of RNAP, and DNA conformational characteristics were examined by use of the alternating copolymer poly(dGdm5C).poly(dGdm5C) as template. Transcription is impeded when the DNA is in the Z conformation as compared with the B; the initial conformation is determined by the concentration of the conformational effectors of Mg2+ and [Co(NH3)6]3+. RNAP binds to both Z and B conformers; the total binding is moderately greater when the template is in the B conformation than when it is strongly stabilized in the Z, by [Co(NH3)6]3+ concentrations much higher than those required for B-Z transition. However, the Z conformer is much more easily displaced competitively from the bulk of its complexes with RNAP than is the B, indicating a specific binding preference for the B conformer. When the template is in the B conformation, or is moderately stabilized in the Z by Mg2+ concentrations such that the polynucleotide is just fully converted from B to Z, elongation is predicted well by chain initiation, indicating that on the Z conformer RNAP is effectively inhibited at the chain initiation or at an earlier stage. The average chain growth rates for polymeric product synthesized on B and on moderately stabilized Z are similar, even though overall RNA synthesis is considerably lowered on the Z form, again indicating that the limiting events precede elongation. When the Z conformer is strongly stabilized, chain initiation and elongation are further inhibited. Elongation is still roughly correlated with chain initiation, but some additional inhibition of elongation takes place independently. Circular dichroism analysis shows that RNAP-DNA binding affects the B-Z conformational equilibrium, leading to reformation of the B conformer from Z and interference with conversion of B to Z, under conditions that would otherwise favor the Z conformer. Thus, there is an RNAP concentration dependent shift of the B-Z transition to higher concentrations of Z-inducing cation, and there is an RNAP concentration dependent decrease in the rate of B to Z conversion. These effects were observed for poly(dGdm5C).poly(dGdm5C), with Z stabilized by [Co(NH3)6]3+ or Mg2+. (They were observed as well for the unmethylated copolymer poly(dGdC).poly(dGdC), with Z stabilized by [Co(NH3)6]3+.) Perturbation of the Z conformer was detectable by circular dichroism at an RNAP:polynucleotide ratio down to a practical limit of approximately 1 RNAP:500 bp.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Selectivity of Escherichia coli RNA polymerase for template conformation. 205 47

Transition from the right-handed B to the left-handed Z conformation of DNA was studied by circular dichroism in parallel with the ability of the DNA to support RNA synthesis with Escherichia coli RNA polymerase. Since the B to Z transition is generally induced by a chemical agent, a definitive demonstration that a change in activity is due to the conformational change, and not to the agent itself or to other factors, requires the clear-cut correlation of template activity and conformation under a variety of conditions that result in conformational change. Such correlation was achieved by following the [Co(NH3)6]3+-induced transition of poly(dG-dC) X poly(dG-dC) and poly(dG-dm5C) X poly(dG-dm5C) and the Mg2+-induced transition of poly(dG-dm5C) X poly(dG-dm5C). In addition, conditions were chosen to minimize possible aggregation. In each of these three systems, the B to Z conformational transition was accompanied by a substantial decrease in transcription activity. While the conversion from B to Z of poly(dG-dm5C) X poly(dG-dm5C) is induced by a 25-fold lower concentration of [Co(NH3)6]3+ than that required for the conversion of unmethylated polymer, in both cases the RNA polymerase activity is decreased at the same cation concentration as that producing the conformational transition. Neither [Co(NH3)6]3+ nor Mg2+ inhibits RNA synthesis with control templates that are not converted to Z under the same conditions, such as poly(dA-dT) X poly(dA-dT) or calf thymus DNA with [Co(NH3)6]3+ or poly(dG-dC) X poly(dG-dC) with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of template conversion from the B to the Z conformation on RNA polymerase activity. 638 35

Double-stranded DNA oligomers were constructed to evaluate the effect of bifunctional and monofunctional platinum(II) complexes at the level of DNA transcription. They contained a single lesion, which is either a cis-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a trans-[Pt(NH3)2(d(GpTpG))] intrastrand cross-link, a cis-[Pt(NH3)2(d(GpC/GpC))] interstrand cross-link, or a (diethylenetriamine)-platinum(II)-dG adduct. The synthetic duplexes were multimerized and then used as templates in dinucleotide-primed reactions catalyzed by prokaryotic or eukaryotic RNA polymerases. Reactions were conducted in the presence of a single triphosphate substrate (single-step addition reaction) or of a combination of triphosphate substrates, permitting elongation of the trinucleotide products to longer RNA chains (productive elongation reaction), respectively. In transcription of the platinated strands, none of the DNA adducts provided an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II. However, the single-step addition reactions were much more impeded from transcription of bifunctional adduct-containing templates as compared to those containing monofunctional lesions. Productive elongation was irreversibly blocked in transcription of the platinated strand of templates containing a cis-d(G*pTpG*) intrastrand cross-link or a cis-d(G*pC/G*pC) interstrand cross-link. In both cases transcription stopped at the level of the lesion. Termination occurred also several nucleotides before the elongation complexes reached the interstrand cross-link. A substantial amount of the RNA polymerase molecules was able of bypassing the trans-d(G*pTpG*) cross-links. In all the cases single-step addition reactions were enhanced on the template strand complementary to that containing the intrastrand cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Spectrum of DNA--platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases. 768 35

Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used to study stalled transcription elongation complexes. In vitro transcription studies were performed in both a promoter-dependent and -independent manner. An elongation complex is strongly blocked by cisplatin 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpTpG) cross-links located on the template strand. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion, the nature of which can be altered by the choice and concentration of NTPs. The [(1R,2R-diaminocyclohexane)Pt]2+ DNA adducts formed by oxaliplatin, which carries a stereochemically more demanding spectator ligand than the ammine groups in cisplatin, also strongly block the polymerase with measurable differences compared with cis-[(NH3)2Pt]2+ lesions. Elongation complexes stopped at sites of platinum damage were isolated and characterized. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. We also discovered that a polymerase stalled at the platinum-DNA lesion can resume transcription after the platinum adduct is chemically removed from the template.
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PMID:Multiple states of stalled T7 RNA polymerase at DNA lesions generated by platinum anticancer agents. 1453

Cationic monofunctional platinum(II)-based anticancer agents with a general formula of cis-[Pt(NH3)2(N-donor)Cl](+) have recently drawn significant attention due to their unique mode of action, distinctive anticancer spectrum, and promising antitumor activity both in vitro and in vivo. Understanding the mechanism of action of novel monofunctional platinum compounds through rational drug design will aid in the further development of active agents. In this study, we synthesized and evaluated a monofunctional platinum-based anticancer agent SA-Pt containing a bulky salicylanilide moiety. The antiproliferative activity of SA-Pt was close to that of cisplatin. Mechanism studies revealed that SA-Pt entered HeLa cells more efficiently than cisplatin, blocked the cell cycle at the S-phase, and induced apoptosis. The compound bound to DNA as effectively as cisplatin, but did not block RNA polymerase II-mediated transcription as strongly as cisplatin, indicating that once the compound formed Pt-DNA lesions, the salicylanilide group was more easily recognized and removed. This study not only enriches the family of monofunctional platinum-based anticancer agents but also guides the design of more potent monofunctional platinum complexes.
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PMID:A monofunctional platinum(II)-based anticancer agent from a salicylanilide derivative: Synthesis, antiproliferative activity, and transcription inhibition. 2545 26